In previous years we had disrupted the genes encoding the pertussis toxin sensitive G proteins Gi2, Gi1, Gi3 and Go. Conditional knockouts for Gi2 and Go were also generated. Double knockouts involving Gi2 and Go are lethal. We expect to learn from combining Gi2 with Gi3 and enhanced survival of Go KO mice by removing the floxed genes at various times after birth. Breeding programs have been set up to add cre recombinase under several specific promoters so as to remove the genes both generally in all issues or in specific cell types such as in dopaminergic neurons to remove Go or lymphocytes to remove Gi from Gi3 KO mice. Most phenotypic studies are done in collaboration with outside investigators, A second aim of this project is to establish the inter-relationship among the Gs, Gi and Gbeta-gamma components of the G protein signaling system, in as much as to learn how they regulate the different adenylyl cyclase (AC) isoforms. Previous studies by others have used recombinant adenylyl cyclases and studied their regulation by purified G proteins activated by non-hydolyzable GTP (GTP-gamma-S). We are using a different approach: we express the different ACs in HEK cells, isolate their membranes - with the properly folded and active enzyme - add to the membranes constitutively active Gs-alpha (GsalphaQ227L)which stimulates all nine AC isoforms (without need of adding a non-hydolyzable GTP) and then study changes in activity due to addition of constitutively active PTX-sentitive G protein alphas (Gi1, Gi2, Gi3, Go1 or Go2),all prepared in myristoylated form as described in our previous Annual Report and do so without and with co-addition of either Gbeta-gamma (prepared in and purified from SF9 cells, or Ca2+/Calmodulin (CaCaM) synthesized in E coli. WE hope inthsi way to establish once and for all whether Go's are inhibitory regulators of ACs and to what extent this may be dependent on an AC subtype. We continued under the guidance of Dr. Yanshun Liu (staff scientist) to work on the co-crystalization of rhodopsin with its cognate G protein transducin. Progress was poor and we terminated this aspect of your activities. We continue collaborating with extramural scientists in the analysis of the phenotypes that arise in G protein deficient mice. The most notable finding this year, in collaboration with Prof. Ahnert-Hilger in Berlin, has been the discovery that the Go2 G protein regulate1d axonal growth. In collaboration with Andrew Tinker in London, we examined and published the relative roles of Gs and Gi in the regulation of the cardiac sinoatrial pacemaker cells. With Prof Bernd Nuernberg in Duesseldorf, and Dr. Mireille Montcouquiol in Bordeaux, we described the role of Gi2 in the asymetric positioning of the primary cilium in hair cells of the inner ear. The fact that Gi2 is required establishes unequivocally that a G protein participates in developmental events that are triggered by non-canonical signaling of Wnt and Hedgehog.